1. Field of the Invention
This invention relates to formulations containing mixtures of oppositely-charged polypeptides such as insulin-like growth factor (IGF-I) and insulin. In particular, this invention entails a formulation containing selected excipients that enable the mixing of oppositely-charged proteins in the same formulation, the excipients preventing the interaction of the proteins that normally would make them precipitate from the solution.
2. Description of Related Art
Human IGF-I is a 7649-dalton polypeptide (Rinderknecht and Humbel, Proc. Natl. Acad. Sci. USA, 73: 2365 (1976); Rinderknecht and Humbel, J. Biol. Chem., 253: 2769 (1978)) belonging to a family of somatomedins with insulin-like and mitogenic biological activities that modulate the action of growth hormone (GH). Van Wyk et al., Recent Prog. Horm. Res., 30: 259 (1974); Binoux, Ann. Endocrinol., 41: 157 (1980); Clemmons and Van Wyk, Handbook Exp. Pharmacol., 57: 161 (1981); Baxter, Adv. Clin. Chem., 25: 49 (1986); U.S. Pat. No. 4,988,675; WO 91/03253; and WO 93/23071. IGF-I contains three disulfide bonds, and has a pI of 8.65 and molar absorptivity of 0.645 at 276 nm. IGF-I naturally occurs in human body fluids, for example, blood and human cerebral spinal fluid. Most tissues and especially the liver produce IGF-I together with specific IGF-binding proteins. Like GH, IGF-I is a potent anabolic protein. See Tanner et al., Acta Endocrinol., 84: 681-696 (1977); Uthne et al., J. Clin. Endocrinol. Metab., 39: 548-554 (1974). See also Ross et al., Intensive Care Med., 19 Suppl. 2: S54-57 (1993), which is a review of the role of insulin, growth hormone, and IGF-I as anabolic agents in the critically ill.
IGF-I may be purified from natural sources, e.g., human serum (Rinderknecht and Humbel, J. Biol. Chem., supra), or made recombinantly (e.g., EP 123,228 and 128,733). Various methods for formulating IGF-I have been described. These include, for example, EP 440,989, which discloses a method for preparing a dried composition of IGF-I, which comprises drying a solution containing IGF-I together with a strong acid, WO 91/18621 on formulating IGF-I in a citrate buffer at pH 6, U.S. Pat. No. 5,374,620 on formulating IGF-I and GH in a growth-promoting composition, U.S. Pat. No. 5,681,814 on formulating IGF-I in an acetate buffer, WO 94/15584 on a stable solution containing IGF-I in a phosphate buffer in an amount of 50 mmol or less, giving a pH of 5.5 to 6.5, which is isotonic and suitable for injection, and WO 95/34318 on a solution comprising IGF-I in an aqueous solution with a reduced concentration of oxygen.
IGF-I has hypoglycemic effects in humans similar to insulin when administered by intravenous bolus injection, but also promotes positive nitrogen balance. Underwood et al., Hormone Research, 24: 166 (1986). IGF-I is known to exert glucose-lowering effects in both normal (Guler et al., N. Engl. J. Med., 317: 137-140 (1987)) and diabetic individuals (Schoenle et al., Diabetologia, 34: 675-679 (1991); Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992)) (see also Sherwin et al., Hormone Research, 41 (Suppl. 2): 97-101 (1994); Takano et al., Endocrinol. Japan, 37: 309-317 (1990); Guler et al., Acta Paediatr. Scand. (Suppl.), 367: 52-54 (1990)), with a time course described as resembling Regular insulin. See also Kerr et al., xe2x80x9cEffect of Insulin-like Growth Factor 1 on the responses to and recognition of hypoglycemia,xe2x80x9d American Diabetes Association (ADA), 52nd Annual Meeting, San Antonio, Tex., Jun. 20-23, 1992, which reported an increased hypoglycemia awareness following rhIGF-I administration. In addition, single administration of rhIGF-I reduces overnight GH levels and insulin requirements in adolescents with IDDM. Cheetham et al., Clin. Endocrinol., 40: 515-522 (1994); Cheetham et al., Diabetologia, 36: 678-681 (1993).
Recombinant human IGF-I administered to Type II diabetics as reported by Schalch et al., J. Clin. Endocrinol. Metab., 77: 1563-1568 (1993) demonstrated a fall in both serum insulin as well as a paralleled decrease in C peptide levels which indicated a reduction in pancreatic insulin secretion after five days of IGF-I treatment. This effect has been independently confirmed by Froesch et al., Horm. Res., 42: 66-71 (1994). In vivo studies in normal rats also illustrate that IGF-I infusion inhibits pancreatic insulin release. Furnsinn et al., Endocrinology, 135: 2144-2149 (1994). In addition, in pancreas perfusion preparations IGF-I also suppresses insulin secretion. Leahy et al., Endocrinology, 126: 1593-1598 (1990). Despite these clear in vivo inhibitory effects of IGF-I on insulin secretion in humans and animals, in vitro studies have not yielded such uniform results.
In vitro studies using multiple concentrations of both IGF-I and glucose have shown various degrees of inhibition of insulin secretion, e.g., from no effect (Sieradzki et al., J. Endocrinol., 117: 59-62 (1988)) to a 30% decrease in insulin release utilizing physiological levels of IGF-I. Van Schravendijk et al., Diabetologia, 33: 649-653 (1990). In a recent study using human pancreatic islets, Eizirik et al., Eur. J. Endocr., 133: 248-250 (1995) found no effect of IGF-I on medium insulin accumulation or on glucose-stimulated insulin release. The investigators speculate that the effect of IGF-I seen in vivo on insulin secretion may be secondary to the extra-pancreatic effects of IGF-I rather than its direct effects on the pancreas. Therefore, the mode and site of action of IGF-I on insulin secretion are not fully understood.
A number of biochemical changes induced by short-term rhIGF-I administration are described in the literature. Prominent among these is a phosphate- and potassium-lowering effect of recombinant human IGF-I (rhIGF-I) reported in healthy subjects during euglycemic clamp. Boulware et al., xe2x80x9cPhosphate and potassium lowering effects of insulin-like growth factor I in humans: comparison with insulinxe2x80x9d The Endocrine Society, 74th Annual Meeting, San Antonio, Tex., 1992June 24-27 See also Guler et al., Acta Paediatr. Scand. (suppl.), 367, supra.
Recombinant human IGF-I (rhIGF-I) has the ability to improve insulin sensitivity. For example, rhIGF-I (70 xcexcg/kg bid) improved insulin sensitivity in non-diabetic, insulin-resistant patients with myotonic dystrophy. Vlachopapadopoulou et al., J. Clin. Endo. Metab., 80: 3715-3723 (1995). Saad et al., Diabetologia, 37. Abstract 40 (1994) reported dose-dependent improvements in insulin sensitivity in adults with obesity and impaired glucose tolerance following 15 days of rhIGF-I treatment (25 xcexcg and 100 xcexcg/kg bid). RhIGF-I also improved insulin sensitivity and glycemic control in some patients with severe type A insulin resistance (Schoenle et al., Diabetologia, 34: 675-679 (1991); Morrow et al., Diabetes, 42 (Suppl.): 269 (1993) (abstract); Kuzuya et al., Diabetes, 42: 696-705 (1993)) or others with non-insulin dependent diabetes mellitus. Schalch et al., xe2x80x9cShort-term metabolic effects of recombinant human insulin-like growth factor I (rhIGF-I) in type II diabetes mellitusxe2x80x9d, in: Spencer EM, ed., Modern Concepts of Insulin-like Growth Factors (New York: Elsevier: 1991) pp. 705-713; Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992).
Though insulin resistance has not been considered a prominent feature of type I diabetes, it is clearly present in some individuals and may be most clinically important during adolescence. As GH has well-known anti-insulin effects, the elevated GH levels during adolescence may mediate much of this insulin resistance. Press et al., supra; Defeo et al., supra; Campbell et al., N. Eng. J. Med., supra, Campbell et al., Metabolism, supra; Arias et al., supra; Davidson et al., supra.
A general scheme for the etiology of some clinical phenotypes that give rise to insulin resistance and the possible effects of administration of IGF-I on selected representative subjects are given in several references. See, e.g., Elahi et al., xe2x80x9cHemodynamic and metabolic responses to human insulin-like growth factor-1 (IGF-I) in men,xe2x80x9d in: Modern Concepts of Insulin-Like Growth Factors, (Spencer, E M, ed.), Elsevier, N.Y., pp. 219-224 (1991); Quin et al., New Engl. J. Med., 323: 1425-1426 (1990); Schalch et al., xe2x80x9cShort-term metabolic effects of recombinant human insulin-like growth factor 1 (rhIGF-I) in type II diabetes mellitus,xe2x80x9d in: Modern Concepts of Insulin-Like Growth Factors, (Spencer, E M, ed.), Elsevier, N.Y., pp. 705-713 (1991); Schoenle et al., Diabetologia, 34: 675-679 (1991); Usala et al., N. Eng. J. Med., 327: 853-857 (1992); Lieberman et al., J. Clin. Endo. Metab., 75: 30-36 (1992); Zenobi et al., J. Clin. Invest., 90: 2234-2241 (1992); Zenobi et al., J. Clin. Invest., 89: 1908-1913 (1992); Kerr et al., J. Clin. Invest., 91: 141-147 (1993). WO 94/16722 discloses a method of chronic modification of cell barrier properties by exposing a cell to a modification-effective amount of IGF-I for at least about seven days and a method of chronic amelioration or reversal of insulin resistance. However, when IGF-I was used to treat type II diabetes patients in the clinic at a dose of 120-160 xcexcg/kg twice daily, the side effects outweighed the benefit of the treatment. Jabri et al., Diabetes, 43: 369-374 (1994). See also Wilton, Acta Paediatr., 383: 137-141 (1992) regarding side effects observed upon treatment of patients with IGF-I.
U.S. Pat. No. 4,988,675 describes treatment of type II diabetics with IGF-I, U.S. Pat. No. 5,466,670 describes treatment of type I diabetics with IGF-I, WO 91/03253 reports use of IGF-I to treat severe insulin-resistant diabetics, and WO 96/01124 describes use of IGF-I to prevent diabetes, delay clinical onset of diabetes, and provide a protective effect against diabetes.
The treatments of choice in type II diabetes have become combination therapies. These combinations historically involved the use of multiple forms of insulin, short-acting insulin, intermediate-acting, and long-acting insulins. Review articles on insulin formulations include Kissel and Volland, Deutsche Apotheker-Zeitunp, 134: 25 (1994) and Campbell, Pharmcy Times. 59: 40 (1993). More recently, combinations of insulin with other anti-diabetic drugs, which are taken orally such as sulphonylureas and biguanides, have become commonplace.
As to combinations of IGF and insulin, Gunn et al., Biochem. Arch., 5: 53-59 (1989) discloses the anabolic effect of insulin and IGF-II. Jacob et al., Am. J. Physiol., 260: E262-E268 (1991) discloses the metabolic effects of IGF-I and insulin in spontaneously diabetic BB/w rats; see also U.S. Pat. No. 4,876,242. Furthermore, the stimulation of cardiac protein synthesis after treatment with insulin and IGF is disclosed by Fuller et al., Biochem. Soc. Trans., 19: 277S (1991). The experiments have been performed in vitro with freshly isolated cardiac myocytes. The effects on protein metabolism after treatment with insulin and IGF on dogs that have been starved overnight are reported by Umpleby et al., Eur. J. Clin. Invest., 24: 337-344 (1994). Shojaee-Moradie et al., J. Clin. Invest., 25: 920-928 (1995) discloses a comparison of the effects of IGF-I, insulin, and combined infusions thereof on glucose metabolism in dogs. Randazzo and Jarett, Exp. Cell Res., 190 (1): 31-39 (1990) discloses characterization of the growth of murine fibroblasts that express human insulin receptors and the effect of IGF-I and insulin on DNA synthesis thereof. Tomas et al., Diabetes, 45: 170-177 (1996) discloses the effects of joint IGF-I and insulin infusion on diabetic rats. Dunger et al., Metabolism, 44: 119-123 (1995) suggests that IGF-I in conjunction with insulin may provide an additional approach to management of IDDM during adolescence. Mathe, Biomedicine and Pharmacotherapy, 49: 221-224 (1995) discloses the role of IGF""s in their relation with insulin for treating diabetes mellitus.
As to the patent literature, U.S. Pat. No. 4,988,675 discloses a combination of IGF-I with a lower amount of insulin than normal to treat Type II diabetes. WO 96/01125 published Jan. 18, 1996 discloses the use of a combination of insulin and an IGF-I in the manufacture of a medicament for counteracting a decrease in nitrogen balance and for counteracting a decrease in protein synthesis and that can be used for treatment of a protein catabolism due to glucocorticoid excess. U.S. Pat. No. 5,091,173 discloses a composition suitable for topical application to mammalian skin or hair comprising a cell-free supernatant from a culture of dermal papilla cells sufficient to increase hair growth comprising one or more members of the IGF family selected from IGF-I, IGF-II, and insulin.
There are various forms of human insulin on the market that differ in the duration of action and onset of action. Jens Brange, Galenics of Insulin, The Physico-chemical and Pharmaceutical Aspects of Insulin and Insulin Preparations (Springer-Verlag, New York, 1987), page 17-40. Regular insulin is a clear neutral solution that contains hexameric insulin. It is short acting, its onset of action occurs in 0.5 hour after injection, and duration of action is about 6-8 hours. NPH (Neutral Protamine Hagedorn)-insulin, also called Isophane Insulin, is a crystal suspension of insulin-protamine complex. These crystals contain approximately 0.9 molecules of protamine and two zinc atoms per insulin hexamer. Dodd et al., Pharmaceutical Research, 12: 60-68 (1995). NPH-insulin is an intermediate-acting insulin; its onset of action occurs in 1.5 hours and its duration of action is 18-26 hours. 70/30 insulin is composed of 70% NPH-insulin and 30% Regular insulin. There are also Semilente insulin (amorphous precipitate of zinc insulin complex), UltraLente insulin (zinc insulin crystal suspension), and Lente insulin (a 3:7 mixture of amorphous and crystalline insulin particles), as well as HUMALOG(copyright) insulin lispro injection (rDNA origin) rapid-acting monomeric insulin solution, as a result of reversing the Lys (B28) and Pro(B29) amino acids on the insulin B-chain) that was recently introduced into the market by Eli Lilly and Company.
NPH-, 70/30, and Regular insulin are the most widely used insulins, accounting for 36%, 28%, and 15%, respectively, of the insulin prescriptions in 1996. These three forms of insulin add up to 79% of all insulin prescriptions. It was therefore determined that the IGF-I formulation needs to be mixable with Regular, NPH-, and 70/30 insulin.
Patients with Type I or Type II diabetes typically take two to four subcutaneous injections of insulin daily to control their blood sugar. The use of an injectable drug other than insulin to treat diabetes, such as IGF-I, is naturally limited due to the desire of diabetics to administer a minimum number of injections. Adding two more subcutaneous injections daily, for IGF-I administration, to regimens that already require several injections per day of insulin is not practical. Further, when combining two proteins such as IGF-I and insulin, it would be necessary to have the resulting formulation stable and well absorbed by the patient, as well as having intermediate-acting insulin. An intermediate-acting insulin regulated in a time- and target-tissue-dependent manner in response to changing demands of the metabolic environment is described by Lewitt et al., Endocrinology, 129: 2254-2256 (1991).
U.S. Pat. No. 5,788,959 discloses a drug-delivery device comprising a single-phase matrix solution of oppositely-charged water-soluble polymers such as polypeptides wherein the matrix solution has dispersed therein a pharmaceutically active ingredient different from the polymers. Further, Burgess et al., J. Pharm. Pharmacol., 43: 232-236 (1991) discloses complex coacervation between oppositely-charged albumin and acacia mixtures, with coacervation being a common method of microencapsulation. Mauk and Mauk, Biochemistry, 21: 4730-4734 (1982) discloses complex formation between purified human methemoglobin and the tryptic fragment of bovine liver cytochrome b5 and report a model for interaction between these molecules in which each hemoglobin subunit binds one cytochrome b5 by means of complementary charge interactions between oppositely-charged groups on the two proteins. Furthermore, EP 615,444 discloses a peroral administration form for peptidic medicaments containing the medicament, such as insulin, distributed in a gelatin or gelatin derivative matrix of opposite charge. EP 313,343 discloses a method of purifying a crude protein from its impurities by ion-exchange chromatography at a pH such that the protein and impurities have an opposite charge so that selective binding occurs.
Presently, diabetics mix NPH-insulin (intermediate-acting neutral protamine hagedorn insulin) with Regular insulin. It would be desirable to mix oppositely-charged polypeptides such as insulin and IGF-I, each from separate vials in the same syringe or other delivery vessel, and to inject or otherwise deliver the mixture immediately. U.S. Pat. No. 5,783,556 discloses a formulation of mixed NPH-insulin and IGF-I. U.S. Pat. No. 5,756,463 discloses a combination of IGF-I and insulin and its use in counteracting a decrease in nitrogen balance and a decrease in protein synthesis. U.S. Pat. No. 4,608,364 discloses an active-compound combination of an insulin derivative and an unmodified insulin or a specific analog thereof for treating diabetes. It would be desirable to mix all types of insulin with IGF-I for this purpose, as well as to mix other polypeptides such as protamine and insulin, which are currently sold as a precipitating complex.
Upon mixing, two polypeptides that are oppositely charged tend to associate with each other and form an aggregate or precipitate out of solution. Use of certain formulation excipients can prevent aggregation and precipitation of such polypeptides when mixed, including IGF-I with insulin and protamine with insulin. Many excipients such as salts, buffers, neutral amino acids, polyols, sugars, and detergents were not effective in preventing precipitation.
Accordingly, the present invention provides, in one embodiment, a composition comprising a mixture of pharmaceutically-active polypeptides of opposite charge and an excipient selected from the group consisting of arginine, lysine, glutamate (glutamic acid), sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine, wherein the polypeptides are soluble in the composition.
In a preferred aspect of this embodiment, the composition may further comprise a buffer such as histidine, at a pH of about 7 to 7.5.
In a further embodiment, the invention provides a kit comprising:
(a) a container comprising the above composition comprising effective amounts of the polypeptides; and
(b) instructions for using the above composition to treat a disorder against which the composition is effective.
If the polypeptides are IGF-I and an insulin, the disorder is preferably a hyperglycemic disorder.
In yet another aspect, the invention supplies a method for preparing the above composition comprising mixing together as component (a) a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, beta-hydroxy cyclodextrin, and beta-cyclodextrin sulfobutyl ether; and as component (b) a second polypeptide having an opposite charge from the first polypeptide.
In a preferred aspect, the method may further comprise the step of incubating the mixture for a period of time at about 30-40xc2x0 C., preferably for about 15 minutes at 37xc2x0 C., wherein the composition further comprises phosph buffered saline. In still another embodiment, the invention provides a kit for preparing the above composition comprising:
(a) a container comprising a first polypeptide in an excipient selected from the group consisting of arginine, lysine, glutamic acid, sodium dodecyl sulfate, and a combination of beta-hydroxy cyclodextrin and arginine;
(b) a container comprising a second polypeptide having an opposite charge from the first polypeptide; and
(c) instructions for combining the contents of containers (a) and (b).
Preferably, the resulting mixture is a pharmaceutically acceptable formulation.
In a still further aspect, the invention provides a method for treating a hyperglycemic disorder such as diabetes in a mammal comprising administering to the mammal, preferably by either injection or infusion, an effective amount of the above composition.
Mixing of two polypeptides of opposite charge such as IGF-I and an insulin requires the physical and chemical properties of both polypeptides to remain unchanged after mixing. Further, it is preferred that the pharmacokinetic and glucose pharmacodynamic profiles remain the same before and after mixing. If one of the polypeptides is IGF-I, it is preferred that the formulation have a minimum of a two-year shelf life. To enable mixing with another polypeptide, the formulation of the first polypeptide can be a clear liquid formulation, or a suspension formulation in which the polypeptide (such as IGF-I) is in the form of crystals, amorphous precipitate, or proteindry powder. An Example herein describes the development of a clear liquid IGF-I formulation that is mixable with insulin.
If the polypeptides are IGF-I and insulin, the targeted IGF-I dose will be 10, 20 or 40 xcexcg/kg/injection (or 1, 2 or 4 xcexcl/kg/injection for a 10 mg/ml IGF-I formulation). From the insulin dosage used by Type I and Type II patients in phase II clinical trials, it can be calculated that the maximum potential mixing ratio can reach 6:1 (vol:vol) for Regular insulin:IGF-I. The minimum potential mixing ratio can reach 1:15 (vol:vol) for NPH-insulin:IGF-I. Therefore, in the Example herein, mixing ratios ranging from 1:6 to 6:1 (vol:vol) were studied for mixing Regular insulin with IGF-I; mixing ratios ranging from 1:15 to 15:1 (vol:vol) were studied for mixing NPH-insulin with IGF-I; and mixing ratios ranging from 1:15 to 6:1 (vol:vol) were studied for mixing 70/30 insulin with IGF-I.